1. Field of the Invention
This invention relates to novel heat and acid-stable alpha-amylase enzymes and processes for producing the same. This invention is also concerned with processes for using these novel alpha-amylase enzymes for hydrolyzing, liquefying and/or converting starch containing materials into starch hydrolysates.
2. Description of the Prior Art
Alpha-amylase enzyme preparations have been used to hydrolyze, liquefy and/or convert starch containing materials into starch hydrolysates as well as being used in detergent formulations. When alpha-amylase enzymes are used to treat starch containing materials, they are used as the initial step in the production of a number of starch hydrolysate materials, such as malto-dextrins, corn syrups, dextrose, levulose, maltose and others. The alpha-amylase enzyme hydrolyzes starch molecules to break them down into a variety of intermediate molecular weight fragments known as malto-dextrins. The malto-dextrins are subsequently treated with one or more additional enzyme preparations including glucoamylase, beta-amylase and glucose isomerase in order to produce the desired final product. Alternatively, a plurality of these enzyme preparations can be introduced into a slurry of the starch material simultaneously to directly produce the desired starch hydrolysate.
Alpha-amylase enzymes are available from a wide variety of sources. Most alpha-amylase enzymes are produced from bacterial sources such as Bacillus subtilis, Bacillus licheniformis, Bacillus stearothermophilus and others which are cultivated in an appropriate culture medium, the cells produced therefrom are then destroyed and the enzyme preparation is thereafter separated from the broth and purified.
Many of the commercially available alpha-amylase enzymes produced today are derived from Bacillus subtilis microorganisms. When these enzymes are used to convert starch to starch hydrolysates, they will generally have an optimal temperature ranging from about 80.degree. to about 85.degree. C., and an optimal pH of about 6.0. The conditions of temperature and pH necessary for efficient use of the enzyme have two disadvantages. Firstly, if starch is converted with the enzyme at a pH of about 6 and at a temperature of about 80.degree. to about 85.degree. C., a part of the reducing end-groups of the starch is isomerized, and in the subsequent conversion process, maltulose is produced which reduces the degree of recovery of the desired product, e.g., dextrose, levulose, or maltose. Secondly, the optimum pH of glucoamylase used in the conversion and saccharification process is generally about 4.5 in the case of Aspergillus niger-type enzymes and a pH of about 5.0 in the case of Rhizopus-type enzymes. Therefore, upon completion of the liquefaction step using the alpha-amylase enzyme, it has been necessary to adjust the pH from about 6 to 4.5 or 5.0. This pH adjustment increases the ion concentration and as a result, increases the load and consequent refining expense using the ion exchange resins used in the purification of the final product.
In recent years, various heat-stable alpha-amylase enzymes have been developed. Examples of such heat-stable alpha-amylase enzymes include those produced from microorganisms derived from Bacillus stearothermophilis as described by Ogasawara et. al., J. Biochem., 67, 65, 77, and 83 (1970); G. B. Manning and L. L. Campbell, J. Biol. Chem., 236, 2952, 2958 and 2962 (1961); S. L. Pfueller and W. H. Elliot, J. Biol. Chem., 244, 48 (1969). More recently, alpha-amylase enzymes having good heat-stability in neutral or weakly alkaline solutions have been made available. These heat and alkaline stable alpha-amylase enzymes have been marketed under the brand name "Thermamyl". They are produced by cultivating microorganisms of the species Bacillus licheniformis as described in British patent specification No. 1,296,839, published Nov. 22, 1972, Madsen et. al., Die Starke, 25, 304, 305 and 308 (1973) and Shigemasa Saito, ABB, 155, 290 (1973). While the alpha-amylase enzymes produced from Bacillus licheniformis have relatively good heat-stability in neutral and weakly alkaline solutions, they do not have suitable stability under acidic conditions to make their use economical from a commercial standpoint.
Accordingly, it is a principal object of the present invention to produce alpha-amylase enzymes which have good heat-stability as well as good stability under acidic conditions, particularly at pH values to render their use under conditions compatible with other amylases such as glucoamylase.